AU2004299755B2 - Method of constructing yellow flower by regulating flavonoid synthesis system - Google Patents

Abstract

There is provided a gene coding for the amino acid sequence listed as SEQ ID NO: 2 or SEQ ID NO: 70, for example. Co-expression of the 4'CGT gene and AS gene in a plant lacking natural aurone synthesis ability is carried out to successfully accumulate aurones and alter the flower color to have a yellow tint. In addition to the expression of both genes, the flavonoid pigment synthesis pathway of the host plant itself is inhibited to obtain flowers with a more defined yellow color.

Description

STY-P952 DESCRIPTION METHOD FOR PRODUCING YELLOW FLOWER BY CONTROLLING FLAVONOID SYNTHETIC PATHWAY 5 Technical Field The present invention relates to a gene coding for a protein having activity of transferring sugars to chalcones, and to plants with modified flower color 10 utilizing the gene. More specifically, the invention relates to a gene coding for a protein having activity of synthesizing chalcone 4'-glucoside, and preferably to a gene derived from the family Scrophulariaceae, and more preferably Antirrhinum majus or Linaria bipartita, coding 15 for a protein having activity of synthesizing chalcone 4'-glucosides, as well as to a method for modifying flower color, and preferably a method for modifying flower color to yellow, by expressing these genes and an aureusidin synthase (hereinafter, "AS") gene either 20 separately or simultaneously and accumulating chalcones or aurones. Background Art Flower color is an important feature for the 25 appreciation and purchasing of ornamental flowers, and flowers with a large variety of colors have traditionally been bred. It is rare for a single species to possess flowers of all colors, as the biosynthesis of pigments that appear as flower colors is genetically determined. 30 Because the gene sources that can be used in hybridization breeding are limited to crossable related varieties, it is essentially impossible to produce flowers of all colors in a target variety by hybridization breeding. Recently, gene recombination 35 techniques have made it possible to obtain flower pigment-synthesizing genes from certain plants and express those genes in different species in order to - 2 achieve modified flower color (Plant Cell Physiol. 39, 1119(1998), Curr. Opin. Biotechnol. 12, 155 (2001)). The flower colors of orange, red, violet and blue are exhibited primarily by flavonoids known as 5 anthocyanins. Yellow colors generally derive from non flavonoid compounds such as carotenoids and betalains, but the yellow colors of some plant species are due to flavonoids. For example, yellow carnations are known to possess 4,21,41,6'-tetrahydroxychalcone (hereinafter, 10 THC) 2'-glucoside in their flower petals (Phytochemistry 5, 111 (1966)). THC 4'-glucoside is also found in Antirrhinum majus and Linaria bipartita. Chalcones such as, THC, butein, isoliquiritigenin and their glycosilated derivatives are known; for 15 example, the aglycon of the glucosides in carnations, morning glory, peony, aster, strawflower, periwinkle, cyclamen and petunia is THC, in Antirrhinum majus (snapdragon) and statice it is 3,4,2',4',6' pentahydroxychalcone (PHC), in cosmos and Jerusalem 20 artichoke it is butein, and in dahlia it is butein and isoliquiritigenin. Also, certain limited species such as snapdragon, Linaria bipartita (toadflax) and morning glory contain yellow flower pigments known as aurones, including aureusidin (hereinafter, AU) and bracteatin. 25 Because the absorption maxima for aurones are between 399 and 403 nm, compared to absorption maxima between 372 and 382 nm for chalcones, their color tones differ and the fluorescence emitted gives aurones a sharper yellow color (Biohorti 1, 49-57 (1990), Seibundo 30 Shinkosha). Chalcones, aurones and anthocyanins usually accumulate as glucosides in the vacuoles of plant cells. The biosynthetic pathway of anthocyanins has been thoroughly studied, and the enzymes involved in anthocyanin synthesis and their coding genes are known 35 (Comprehensive Natural Products Chemistry, vol I (ed. Sankawa) pp713-748, Elsevier, Amsterdam (1999)). The biosynthetic pathway of flavonoids is widely N:\Melbourne\Cases\Patent\61000-61999\P61155.AU\Specis\Speci.doc 5 July 2007 - 3 distributed among the higher plants and is conserved among species. THC is biosynthesized from three molecules of malonyl CoA and one molecule of coumaroyl CoA, by the catalytic action of chalcone synthase. THC 5 exhibits a light yellow color, but in plant cells it is usually rapidly converted to colorless naringenin by chalcone isomerase (CHI). Also, THC is highly unstable at near neutral pH and is converted to naringenin by spontaneous ring closure. For THC to exist stably in 10 plant cells, i.e. for it to stably exhibit a yellow color, the 2'-position of THC must be modified with a saccharide to prevent its ring closure. The reaction is catalyzed by an enzyme that transfers glucose to the 2' position of THC (UDP-glucose: 4,2',4',6' 15 tetrahydroxychalcone 2'-glucosyltransferase, hereinafter abbreviated as 2'CGT). THC 2'-glucoside is present in carnation and cyclamen, and therefore 2'CGT is also predicted to be found in their flowers. Thus, it was conjectured that if 20 the 2'CGT gene could be obtained and the enzyme gene expressed in a plant, it should be possible to accumulate THC 2'-glucosides and produce yellow flowers (Biotechnology of Ornamental Plants, Edited by Geneve, Preece and Merkle, pp259-294, CAB International 25 Wallingford, UK (1997)). Moreover, it was discovered that adequately accumulating THC 2'-glucoside and exhibiting yellow color requires deletion of the CHI gene to suppress enzymatic conversion from THC to naringenin, and that a clear yellow color also requires deletion of 30 the gene for flavanone 3-hydroxylase (hereinafter abbreviated as F3H) in addition to the CHI gene (Plant Cell Physiol. 43, 578 (2002)). While cloning of the carnation 2'CGT gene has been reported to date (Plant Cell Physiol. 44, s158 (2003)), 35 its sequence has not been published. Also, the gene coding for 2'CGT activity has been obtained from carnation and expressed in petunia, thereby accumulating -4 THC 2'-glucoside in petunia petals (PCT/JP03/10500). However, the THC 2'-glucoside produced by 2'CGT does not have a chemicalstructure that can serve as a precursor for aurone synthesis. Also, as mentioned above, 5 accumulation of THC 2'-glucoside results in only light yellow petals. It is known that faint yellow petals are produced by accumulation of THC having the 2'-hydroxyl methylated, but the nature of the enzyme that catalyzes this 10 methylation and of its gene is unknown. Yellow varieties such as dahlia and cosmos contain 6'-deoxychalcone. In legumes, 6'-deoxychalcone is the precursor of 5 deoxyflavonoid, which is synthesized by the catalytic action of chalcone synthase (CHS) and chalcone reductase 15 (CHR). It has been reported that introduction of the alfalfa CHR gene into petunia produced 6'-deoxychalcones such as butein, and that when the CHR gene was introduced into white flower petunia the flowers were mostly white upon blooming although a very light yellow color was 20 observed at the budding stage, and therefore it was not possible to create an industrially useful yellow flower (Plant J. 13, 259 (1998)). Because aurones exhibit a more brilliant yellow color than chalcone glucosides as explained above, it 25 would be highly useful, for the industry, to develop a method for accumulating aurones. AS, one of the enzymes involved in aurone biosynthesis, and its gene, have already been reported in the literature (Science, 290, 1163 (2000)). According to this report, AS produces AU, 30 bracteatin and their glucosides from THC, PHC and their glucosides as substrates. However, use of the AS gene to produce and accumulate aurones such as AU and bracteatin has not been described. The present inventors have constructed a binary 35 vector having the AS gene linked downstream from a structural promoter and introduced the AS gene into petunia and torenia by the Agrobacterium method, but no - 5 accumulation of aurones was observed. It has also been reported that 3-glucosylation of anthocyanidin is essential for transportation of anthocyanins into vacuoles (Nature 375, 397 (1995)), suggesting that 5 glucosylation of aurones is likewise necessary as a transportation signal into vacuoles. In fact, the major aurone that accumulates in yellow Antirrhinum majus flower petals is AU 6'-glucoside. A GT exhibiting AU 6' glucosylating activity (AU6GT) (WO 00/49155) was 10 therefore obtained, and the AU6GT gene was constitutively expressed in petunia together with the AS gene, but accumulation of aurones was not observed. The enzymes involved in flavonoid and anthocyanin biosynthesis are believed to localize in the cytoplasm or 15 endoplasmic reticulum of cells. By the actions of these enzymes, flavonoids and anthocyanins are synthesized and glucosylated outside of vacuoles, i.e. in the cytoplasm, and transported into the vacuoles (Natural Product Reports 20, 288, (2003)). However, ardent research led 20 the present inventors to the finding that AS is exceptional in that it localizes in the vacuoles. Thus, it was hypothesized that glucosylated chalcones may be transported to the vacuoles in vivo and used as substrates for synthesis of aurones in the vacuoles. 25 As mentioned above, AU 6'-glucoside is the major aurone that accumulates in the vacuoles of yellow Antirrhinum majus petals. The 6' position of AU corresponds to the 4' position of THC, and THC 4' glucosides are also present in yellow Antirrhinum majus 30 petals. On this basis, an aurone synthetic pathway was inferred wherein glucosylation of the 4' position of THC synthesized in the cytoplasm is followed by transport to the vacuoles, and this is used as substrate for synthesis of AU 6'-glucosides by AS. Thus, it was concluded that 35 synthesis of THC 4'-glucoside is essential for synthesis of aurones such as AU 6'-glucoside in different plant varieties. For this purpose, UDP-glucose:4,2',4',6'- - 6 tetrahydroxychalcone 4'-glucosyltransferase (hereinafter, 4'CGT) is required for 4'-glucosylation of THC, and therefore the 4'CGT gene must be obtained. However, cloning of the 4'CGT gene has not yet been reported, nor 5 do reports exist of isolating 4'CGT. Enzymes that catalyze glucosylation of a variety of compounds including flavonoids to produce glucosides are generally referred to as glucosyltransferases (GT), and plants possess a large diversity of GT molecules and 10 their coding genes, corresponding to the types of substrates and transferred sugars. Because GT enzymes usually utilize UDP-glucose as the glucose donor, they contain in their amino acid sequence a motif that binds UDP-glucose (Plant J. 19, 509 (1999)). Already, GT genes 15 carrying this motif are known in 99 species of Arabidopsis whose entire genome structure has been elucidated (J. Biol. Chem. 276, 4338, (2001)). GT enzymes and amino acid sequences and functions have also been worked out in several other plants. The 20 genes for enzymes catalyzing reactions of transferring sugars to the 3-hydroxyl groups of flavonoids or anthocyanidins (UDP-glucose:flavonoid 3 glucosyltransferase, hereinafter: 3GT) have been obtained from perilla, corn, gentian, grape and the like (J. Biol. 25 Chem. 274 , 7405 (1999); J. Biol. Chem. 276, 4338, (2001)). In addition, genes for enzymes catalyzing reactions of transferring sugars to the 5-hydroxyl groups of anthocyanins (UDP-glucose:anthocyanin 5 glucosyltransferase, hereinafter: 5GT) have been obtained 30 from perilla, verbena and the like (J. Biol. Chem., 274, 7405, (1999)). Analysis of the amino acid sequences of 3GT and 5GT has shown that GT enzymes with the same function have similar amino acid sequences even in different plant 35 varieties, or in other words, that they constitute a family (J. Biol. Chem. 276, 4338, (2001)). Thus, it is not difficult to obtain enzymes having the same function N:\Melbourne\Cases\Patent\61000-61999\P61155.AU\Specis\Speci.doc 5 July 2007 as known GT enzymes (i.e., orthologs) from other plant varieties, given the current level of technology. For example, the petunia 5GT gene has been cloned using the perilla 5GT gene (Plant Mol Biol. 48, 401 (2002)). 5 However, much laborious trial and error is required to obtain a novel GT gene having absolutely no known ortholog. As regards Arabidopsis whose entire genome structure is known as mentioned above, its flower petals are white 10 and accumulation of chalcone 4'-glucosides has not been reported. Consequently, Arabidopsis GT gene information cannot be used for cloning of the 4'CGT gene. Moreover, even though 2'CGT has been isolated from carnation (PCT/JP03/10500), high homology does not necessarily 15 exist between the 4'CGT gene and the 2'CGT gene. This is because the biochemical and molecular biological features of each GT may differ substantially if the position of sugar addition is different, even if the substrate is the same. This is also supported by the fact that 3GT and 20 5GT belong to different GT families. Also, betanidine 5GT and 6GT have the same substrates, and yet their amino acid homology has been reported to be only 19% (Planta 214, 492 (2002)). In fact, the GT enzymes that transfer sugars to the 25 3-, 5- and 3'-positions of the same anthocyanidin skeleton belong to different families in the GT superfamily, and the amino acid homology between these families is no more than about 20% (Plant Physiol. 132, 1652, (2003), Natural Product Reports 20, 288, (2003)). 30 Several methods are possible for obtaining not only the 4'CGT gene but also novel genes. For example, the genes for enzymes expressed in flower petals, which are involved in the synthesis of rose scent components, have been extensively sequenced and have been identified by 35 their structures, expression patterns and expression in E. coli (Plant Cell. 14, 2325 (2002)). In order to identify the 4'CGT gene, 5,000 clones were randomly -8 selected from a cDNA library derived from the petals of yellow Antirrhinum majus (Butterfly Yellow variety) which accumulates aurone and chalcone 4'-glucosides, and their nucleotide sequences were determined. 5 As a result of homology search using a public DNA database, three different GT genes were obtained. Two of the genes were the 3GT gene and the aforementioned AU6GT coding gene (WO 00/49155), while the remaining gene was for a novel GT (designated as pSPB662) (SEQ ID NO: 13). 10 However, the GT encoded by pSPB662 exhibited no glucosylating activity for THC, and was clearly not 4'CGT. Furthermore, as mentioned above, high expression of this gene together with the AS gene in petunia resulted in no observable production of chalcone 15 glucosides or aurones, nor was any change in flower color seen. These results suggest that a chalcone glucosylating enzyme gene cannot be isolated by random screening of approximately 5,000 clones, and it was therefore difficult to obtain a 4'CGT gene. 20 Patent document 1: PCT/JP03/10500 Patent document 2: WO 00/49155 Non-patent document 1: Plant Cell Physiol. 39,1119 (1998) Non-patent document 2: Curr. Opin. Biotechnol. 12, 25 155 (2001) Non-patent document 3: Phytochemistry 5,111 (1966) Non-patent document 4: Biohorti 1 49-57 (1990) SEIBUNDO SHINKOSHA Non-patent document 5: Comprehensive Natural 30 Products Chemistry, vol I (ed. Sankawa) pp7'13-748, Elsevier, Amsterdam (1999) Non-patent document 6: Biotechnology of Ornamental Plants, Edited by Geneve, Preece and Merkle, pp259-294, CAB International Wallingford, UK (1997) 35 Non-patent document 7: Plant Cell Physiol. 43, 578 (2002) Non-patent document 8: Plant Cell Physiol. 44, s158 N.\Melbourne\Cases\Patent\61000-61999\P61155.AU\Specis\Speci.doc 5 July 2007 -9 (2003) Non-patent document 9: Plant J. 13, 259 (1998) Non-patent document 10: Science, 290, 1163 (2000) Non-patent document 11: Nature 375, 397 (1995) 5 Non-patent document 12: Natural Product Reports 20, 288, (2003) Non-patent document 13: Plant J. 19, 509 (1999) Non-patent document 14: J. Biol. Chem. 276, 4338, (2001) 10 Non-patent document 15: J. Biol. Chem. 274, 7405 (1999) Non-patent document 16: Plant Mol Biol. 48, 401 (2002) Non-patent document 17: Planta 214, 492 (2002) 15 Non-patent document 18: Plant Physiol. 132, 1652, 2003 (2003) Non-patent document 19: Plant Cell. 14, 2325 (2002) All references, including any patents or patent applications, cited in this specification are hereby 20 incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be 25 clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. 30 Disclosure of the Invention In a first aspect, the invention provides an isolated gene coding for a protein having activity of transferring a sugar to the chalcone 4'-position, wherein 35 said gene is selected from the group consisting of: (i) a gene coding for the amino acid sequence listed as SEQ ID NO: 2 or SEQ ID NO: 70; 10348941 (GHMatters) 28-Oct-09 10 (ii) a gene which codes for a protein having the amino acid sequence listed as SEQ ID NO: 2 or SEQ ID NO: 70 with a modification of one or a plurality of amino acids that are added, deleted and/or substituted with 5 other amino acids, and having activity of transferring a sugar to the chalcone 4'-position; and (iii) a gene which hybridizes to all or a portion of the nucleotide sequence listed as SEQ ID NO: 1 or SEQ ID NO: 69 under stringent conditions and codes for a protein 10 having activity of transferring a sugar to the chalcone 4'-position. Disclosed herein is a protein having activity of transferring sugars to the 4'-hydroxyl groups of chalcones, and its gene, and preferably a protein having 15 activity of transferring a sugar to the 4'-hydroxyl groups of chalcones specifically, and its gene. Also disclosed are plants with modified flower color, preferably altered to yellow, using the aforementioned GT gene. 20 As mentioned above, the biochemical and molecular biological features of 4'CGT are unknown, and the enzyme has not been purified nor its gene cloned. The present inventors, using a probe having a nucleotide sequence corresponding to the conserved amino acid sequence of the 25 GT family, obtained ten different GT genes having the nucleotide sequence for the conserved amino acid sequence from a yellow Antirrhinum majus (Butterfly Yellow) flower petal cDNA library. The GT gene group was expressed in different E. coli cells, activity of transferring glucose 30 to the chalcone 4'-position, i.e. 4'CGT activity, was confirmed among the E. coli extracts, and it was confirmed that the cloned gene coded for 4'CGT. The gene was expressed in plants for modification of flower color, and the present invention was thereupon completed. 35 Specifically, the invention relates to (1) a gene coding for a protein having activity of transferring a sugar to the chalcone 4'-position. 1034894_1 (GHMatters) 28-Oct-09 11 The invention further relates to (2) a gene according to (1) above coding for the amino acid sequence listed as SEQ ID NO: 2. The invention further relates to (3) a gene 5 according to (1) above, which hybridizes to all or a portion of the nucleotide sequence listed as SEQ ID NO: 1 under conditions of 5 x SSC, 50*C and codes for a protein having activity of transferring a sugar to the chalcone 4'-position. 10 Disclosed herein is (4) a gene according to (1) above, which codes for a protein having the amino acid sequence listed as SEQ ID NO: 2 with a modification of one or a plurality of amino acids that are added, deleted and/or substituted with other amino acids, and having 15 activity of transferring a sugar to the chalcone 4' position. Also disclosed is (5) a gene according to (1) above, which hybridizes to DNA comprising all or a portion of the nucleotide sequence listed as SEQ ID NO: 1 under 20 stringent conditions and codes for a protein having activity of transferring a sugar to the chalcone 4' position. In one embodiment, a gene according to the first aspect is derived from the family Scrophulariaceae. 25 In a second aspect, the invention still further provides a vector comprising a gene according to the first aspect. In a third aspect, the invention still further provides an isolated host cell transformed by a vector 30 according to the second aspect. In a fourth aspect, the invention still further provides a protein encoded by a gene according to the first aspect. In a fifth aspect, the invention still further 35 provides a method of producing a protein having activity of transferring a sugar to the chalcone 4'-position, comprising culturing or growing a host cell according to 1034894_1 (GHMatters) 28-Oct-09 12 the third aspect and obtaining the protein from the host cell. In a sixth aspect, the invention still further provides a plant comprising a gene according to the first 5 aspect introduced therein or a progeny of the plant having the same properties as the plant, or a vegetatively propagated plant or tissue from such a plant. In a seventh aspect, the invention still further 10 provides a flower cut from a plant according to the sixth aspect. In an eighth aspect, the invention still further provides a method for transferring a sugar to the chalcone 4'-position using a gene according to the first 15 aspect. In a ninth aspect, the invention still further provides a plant comprising modified flower color obtained by introducing and expressing a gene according to the first aspect into the plant, or a progeny of the 20 plant having the same properties as the plant. In an embodiment of the ninth aspect, the invention still further provides a plant wherein the flower color has a yellow tint. In a tenth aspect, the invention still further 25 provides a method of altering a flower color to yellow, comprising the steps of introducing into a plant a gene according to the first aspect together with a gene coding for aureusidin synthase and expressing the genes in the plant. 30 In an eleventh aspect, the invention still further provides a method of altering a flower color to yellow, comprising the steps of introducing into a plant a gene according to the first aspect together with a gene coding for aureusidin synthase, and expressing the genes in the 35 plant, while also inhibiting expression of a flavonoid synthesis pathway gene in the plant. In a twelfth aspect, the invention still further 1034894_1 (GHMatters) 28-Oct-09 - 12a provides a method of altering a flower color to yellow, comprising the steps of introducing into a plant a gene according to the first aspect together with a gene coding for aureusidin synthase, and expressing the genes in the 5 plant, while also inhibiting expression of dihydroflavonol reductase gene in the plant. In a thirteenth aspect, the invention still further provides a method of altering a flower color to yellow, comprising the steps of introducing into a plant a gene 10 according to the first aspect together with a gene coding for aureusidin synthase, and expressing the genes in the plant, while also inhibiting expression of flavanone 3 hydroxylase gene in the plant. In the claims which follow and in the description of 15 the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated 20 features but not to preclude the presence or addition of further features in various embodiments of the invention. Brief Explanation of the Drawings Fig. 1 shows the typical flavonoid synthesis pathway 25 in a plant. The details of the metabolic pathway differ for different plant species, depending on the presence or absence of the enzyme genes shown. For example, yellow Antirrhinum majus petals have both the anthocyanin synthesis pathway and pathway that leads to aurone 30 synthesis, whereas torenia which is a plant belonging to the family Scrophulariaceae lacks the 4'CGT and AS genes and therefore cannot synthesize aurones. The abbreviations used in the drawing are explained below. CHS = chalcone synthase; CHI = chalcone isomerase; F3H = 35 flavanone 3-hydroxylase; DFR = dihydroflavonol 4 reductase; ANS = anthocyanidin synthase; 3GT = UDP glucose:anthocyanidin 3-glucosylase; FLS = flavonol 1034894_ 1 (GHMatters) 28-Oct-09 - 12b synthase; FNS = flavone synthase; F3'H = flavonoid 3' hydroxylase; F3',5'H = flavonoid 3',5'-hydroxylase; 2'CGT = UDP-glucose:4,2',4',6'-tetrahydroxychalcone 2' glucosyltransferase; 4'CGT = UDP-glucose:4,2',4',6' 5 tetrahydroxychalcone 4'-glucosyltransferase; AS = aureusidin synthase. Fig. 2 is a continuation of Fig. 1. Fig. 3 shows the results of Southern hybridization 1034894_1 (GHMatters) 28-Oct-09 - 13 of a torenia transformant. Genomic DNA was extracted from pSFL201, pSFL307 and pSFL308 introduced torenia leaves (variety Summerwave Blue) transformants, and after KpnI cleavage, was subjected to Southern hybridization 5 using the 4'CGT gene as the probe. The numerals at the top of each lane indicate the lineage numbers of the transgenic constructs and transformants. SWB represents Summerwave Blue used as the host. M1 and M2 are DIG labeled size markers (Roche), and the size of each band 10 is shown at left. Best Mode for Carrying Out the Invention As an example of a gene according to the invention there may be mentioned one coding for the amino acid 15 sequence listed as SEQ ID NO: 2. However, it is well known that a protein having a modified amino acid sequence with one or a plurality of amino acids added, deleted and/or substituted with other amino acids can retain the enzyme activity of the original protein. The 20 present invention therefore also includes any protein having the amino acid sequence listed as SEQ ID NO: 2 with a modification of one or a plurality of amino acids that are added, deleted and/or substituted with other amino acids, so long as it is a protein that retains 25 4'CGT activity, as well as a gene coding for such a protein. Here, "a plurality" means 2-30, and preferably 2-9. The present invention also relates to a gene that hybridizes to DNA having the nucleotide sequence listed 30 as SEQ ID NO: 1 under the relatively mild conditions of 5xSSC, 50*C, and that codes for a protein having 4'CGT activity. Also included within the technical scope of the invention is any gene that hybridizes to DNA having the nucleotide sequence listed as SEQ ID NO: 1 under 35 stringent conditions, and that codes for a protein having 4'CGT activity. Here, the "stringent conditions" may be, for example, 2xSSC, 65*C, but the hybridization conditions - 14 are not limited thereto because they may differ depending on the length and base composition of the DNA used as probe. As genes selected by such hybridization there may be 5 mentioned naturally occurring genes such as plant-derived genes, preferably Scrophulariaceae-derived genes, and more preferably Antirrhinum majus, Linaria bipartita and Linaria japonica-derived genes, although there is no limitation to plants. That is, the 4'CGT gene of the 10 invention is not limited to the 4'CGT gene from Antirrhinum majus, Linaria bipartita or Linaria japonica, and the 4'CGT gene from other biological species that contain chalcone 4'-glucosides may be used to cultivate yellow-colored flowers. Synthetic DNA containing the 15 4'CGT gene may also be used in the same manner as plant derived genes. The gene selected by hybridization may be cDNA or genomic DNA. As shown in the examples, a gene having homology 20 with the conserved region of GT may be obtained by screening a cDNA library prepared from, for example, Antirrhinum majus or Linaria bipartita flower petals. DNA coding for GT having an amino acid sequence which is a modification of the amino acid sequence listed as SEQ 25 ID NO: 2 may be synthesized by a publicly known site specific mutagenesis or PCR method using DNA having the nucleotide sequence listed as SEQ ID NO: 1. For example, a DNA fragment for amino acid sequence modification may be obtained by restriction endonuclease treatment of cDNA 30 or genomic DNA and used as template for site specific mutagenesis or PCR using a primer corresponding to the desired modified amino acid sequence, in order to obtain a DNA fragment corresponding to the desired modified amino acid sequence. The modification-introduced DNA 35 fragment may then be ligated to a DNA fragment coding for another portion of the enzyme of interest. Such DNA may also be chemically synthesized.

- 15 Alternatively, in order to obtain DNA coding for a protein comprising a shortened amino acid sequence, DNA coding for an amino acid sequence longer than the target amino acid sequence, for example, the full-length amino 5 acid sequence, may be cleaved with a desired restriction endonuclease, and if the resulting DNA fragment does not code for the full-length target amino acid sequence, it may be ligated with a synthesized DNA fragment corresponding to the lacking portion of the amino acid 10 sequence. The GT gene obtained in this manner is expressed using a gene expression system in E. coli or yeast, and measurement of 4'CGT activity in the E. coli or yeast extract can confirm that the obtained GT gene codes for a 15 protein exhibiting 4'CGT activity. The 4'CGT activity may be measured in the manner described in Example 3, for example, by adsorbing a chalcone serving as the substrate for 4'CGT onto a reverse-phase resin and then reacting the reverse-phase resin with extract of E. coli or yeast 20 that has been transformed with the GT gene, and analyzing the produced chalcone 4'-glucoside by high performance liquid chromatography (HPLC). The obtained 4'CGT gene may be expressed in suitable host cells to obtain 4'CGT protein as the gene product. 25 Alternatively, an antibody for a protein or peptide having all or a portion of the amino acid sequence listed as SEQ ID NO: 2 may be used to obtain the 4'CGT gene of another organism by expression cloning. The present invention further relates to a 30 recombinant vector, especially an expression vector, containing the 4'CGT gene, and to host cells transformed using the vector. The host cells used may be prokaryotic or eukaryotic. As examples of prokaryotic cells there may be used publicly known host cells including bacteria 35 belonging to the genus Escherichia such as Escherichia coli, or microorganisms belonging to the genus Bacillus such as Bacillus subtilis.

- 16 As examples of eukaryotic cells there may be used eukaryotic microorganisms, and preferably yeast or filamentous fungi. As examples of yeast there may be mentioned Saccharomyces yeast such as Saccharomyces 5 cerevisiae, and as examples of filamentous fungi there may be mentioned Aspergillus microbes such as Aspergillus oryzae and Aspergillus niger, or Penicillium microbes. Animal or plant cells may also be used as host cells, among which mouse, hamster, monkey or human cell lines 10 may be used as the host cells. Insect cells such as silkworm cells, or actual silkworm adults, may be used as hosts. The expression vector of the invention includes expression regulating regions such as a promoter and 15 terminator dependent on the host species, as well as a replication origin. As promoters for the expression vector in bacteria, and particular in E. coli, there may be used conventional publicly known promoters such as trc promoter, tac promoter and lac promoter. As yeast 20 promoters there may be used, for example, glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter or the like and as filamentous fungi promoters there may be used, for example, amylase or trpC promoters, but there is no limitation to these promoters. As animal cell 25 promoters there may be used viral promoters, such as SV40 early promoter or SV40 late promoter. The expression vector may be constructed by ordinary methods using restriction endonucleases, ligases and the like. Transformation of the host cells with the expression 30 vector may also be carried out by conventional publicly known procedures. Construction of a plant expression vector may be accomplished, for example using a binary vector such as pBIl21 when using Agrobacterium, or using an E. coli 35 vector such as pUC19 when using a particle gun. The plant cells transformed with the plant expression vector may then be selected using a marker gene such as an - 17 antibiotic resistance gene and regenerated under conditions employing a suitable plant hormone, to obtain transformed plant individuals having the 4'CGT gene introduced therein. Cultivation of the transformed 5 plants to blooming can yield plants exhibiting the modified flower color. The host cells transformed by the expression vector or the transformed plants may be cultured or cultivated, and the target 4'CGT protein recovered and purified from 10 the culture, etc. by ordinary methods such as, for example, filtration, centrifugal separation, cell disruption, gel filtration chromatography, ion-exchange chromatography and the like. The present invention is not limited only to the 15 4'CGT gene of Antirrhinum majus or Linaria bipartita, but may also be applied for modification of flower color by 4'CGT whether the source of the 4'CGT or 4'CGT gene is a plant, animal, microorganism or synthesized organism. The present invention relates to the use of the 4'CGT 20 gene, and the scope of the invention encompasses plants with modified flower color by introduction and expression of the 4'CGT gene in plants, as well as their progeny, vegitatively propagated products of the foregoing and tissues of such plants, where the tissues may include cut 25 flowers. Moreover, the scope of the present invention further encompasses plants with modified flower color by introduction and expression in plants not only of the 4'CGT gene but even AS genes other than the 4'CGT gene, and by inhibited expression of the endogenous flavonoid 30 synthesis pathway genes of the host, as well as their progeny, vegitatively propagated products of the foregoing and tissues of such plants, where the tissues may include cut flowers. With the current level of technology it is possible 35 to introduce genes into plants and express those genes either constitutively or in a tissue-specific manner, or to inhibit expression of target genes by antisense, - 18 cosuppression or RNAi methods. As examples of plants to be transformed there may be mentioned rose, chrysanthemum, carnation, snapdragon, cyclamen, morning glory, begonia, impatiens, geranium, orchid, bluebell, 5 freesia, gerbera, gladiolus, gypsophila, kalanchoe, lily, pelargonium, geranium, petunia, torenia, tulip, rice, forsythia, begonia, barley, wheat, rapeseed, potato, tomato, poplar, banana, eucalyptus, sweet potato, soybean, alfalfa, lupin, corn and cauliflower, although 10 there is no limitation to these. Examples The present invention will now be explained in greater detail by the following examples. The molecular 15 biological methods employed were based on the description in W096/25500 or Molecular Cloning (Sambrook et al. Cold Spring Harbour Laboratory Press, 1989), unless otherwise specified. Example 1. Construction of yellow Antirrhinum majus 20 flower petal cDNA library A cDNA library was constructed as described in the literature (Science 290, 1163 (2000)), from 5 g of fresh flower petals of the yellow Antirrhinum majus variety Butterfly Yellow. The obtained library consisted of 1.6 25 x 101 plaque forming units. Example 2. 4'CGT gene screening 1 Previously disclosed GT amino acid sequences were compared, and nucleotide sequences corresponding to the conserved regions of the amino acid sequences were 30 amplified and used as probes for screening of the Antirrhinum majus cDNA library described in Example 1. Five GTs were used as the probes, namely the sequences for morning glory-derived UDP glucose:anthocyanidin 3-glucoside glucosyltransferase 35 (3GGT) (Japanese Unexamined Patent Publication No. 2003 289884), petunia-derived 3GT (Plant Mol. Biol. 48, 401, (2002)), verbena-derived 5GT (J. Biol. Chem. 274, 7405 - 19 (1999)), Scutellaria baicalensis-derived GT (SBGT, Planta 210,1006 (2000)) and gentian-derived UDP glucose:anthocyanin 3'-glucosyltransferase (3'GT) (Plant Physiol. 132,1652, (2003)). One oligonucleotide pair was 5 synthesized for each GT to allow amplification of the conserved region sequence. The oligonucleotide sequences are listed as SEQ ID NO: 3-12. Morning glory 3GGT SEQ ID NO: 3: 5'-GAA ATG GTC GGA TTG GCT GGG-3' 10 SEQ ID NO: 4: 5'-ACC TCC ACC CCA ACT TTC AGG-3' Petunia 3GT SEQ ID NO: 5: 5'-GAT GCA TAA TTT GGC TAG AAA AGC-3' SEQ ID NO: 6: 5'-CCA ATT TGC CAA ACA CTT TCC-3' Verbena 5GT 15 SEQ ID NO: 7: 5'-TGC CTC GAA TGG TTG AGC ACG-3' SEQ ID NO: 8: 5'-CTC TCA CTC TCA CAC CCG-3' Baikal skullcap GT SEQ ID NO: 9: 5'-CAC GAA TGC TTA GCA TGG CTC-3' SEQ ID NO: 10: 5'-CTT ATT GCC CAC TGA AAC CCC-3' 20 Gentian 3'GT SEQ ID NO: 11: 5'-TGT CTG AAT TGG CTT GAT TCC-3' SEQ ID NO: 12: 5'-AAC CCA CAG AAA CCC CTG TTC-3' The probes were labeled using a non-radioisotope DIG-nucleic acid detection system (Roche Diagnostic 25 Corp.), with PCR under the conditions recommended by the manufacturer. Plasmids containing 1 ng of each cDNA were used as template and 100 ng of oligonucleotide specific to each gene was used as primer, for 25 cycles of PCR where each cycle consisted of reaction at 95*C for 1 30 minute, 550C for 1 minute and 720C for 2 minutes. An equivolume mixture of the PCR amplification product for each gene was used as the hybridization probe for screening of the Antirrhinum majus cDNA library of Example 1. 35 The hybridization was carried out overnight at 37*C in 5XSSC containing 30% formaldehyde, 1% SDS, and the - 20 filter was rinsed for 30 minutes at 550C using 5XSSC and 1% SDS. The positive signal by screening was detected using a non-radioisotope DIG-nucleic acid detection system (Roche Diagnostic Corp.), according to the method 5 recommended by the manufacturer. Approximately 300,000 plaques were screened, leading to selection of 10 clones possessing full-length glucosyltransferase genes, which were designated as pSPB264, 1621, 1620, 1622, 1610, 1609, 1617, 1615, 660 and 658. A DNA Sequencer model 3100 10 (Applied Biosystems) was used to determine their cDNA sequences by the primer walking method with synthetic oligonucleotide primers. The nucleotide sequences of the amino acid-coding regions of the cDNA are listed as SEQ ID NO: 14-23. 15 Example 3. Assay of chalcone GT activity using E. coli 3-1 Construction of E. coli expression vector and expression of GT in E. coli The activity of GT encoded by the ten cDNAs obtained 20 in Example 2 was analyzed using an E. coli expression system. First, E. coli expression constructs were prepared for each cDNA. An NcoI site was introduced by PCR so as to be situated on the nucleotide sequence ATG believed to be the start codon for each cDNA, and the 25 region from the start methionine to the end codon was linked to the NcoI and KpnI or NcoI and EcoRV sites of the E. coli expression vector pQE61 (QIAGEN). The PCR solution (25 pl) for introduction of the NcoI site situated on the start methionine comprised each 30 GT cDNA as template, 0.2 pmol/pl each of primer containing the NcoI recognition site situated on the start methionine position and primer from the 3' end to the 5' end near the stop codon, lx ExTaq buffer (Takara), 0.2mM dNTPs and 1.25 U ExTaq polymerase. Reaction was 35 conducted at 94*C for 5 minutes, followed by 28 cycles of reaction at 940C for 1 minute, 55*C for 1 minute and 72 0

C

- 21 for 2 minutes, and then by final treatment at 72 0 C for 5 minutes. The obtained PCR product was subcloned in pCR2.1 TOPO vector (INVITROGEN) according to the method recommended by the manufacturer. The DNA sequences of 5 the amplified DNA fragments were analyzed, and after confirming lack of PCR-induced error, they were introduced into E. coli expression vector pQE61 (QIAGEN). For example, for cDNA encoded in pSPB1617 (SEQ ID NO: 20), the two different primers 1617 BamHINcoI-FW (SEQ 10 ID NO: 24) and the 1617 XhoIKpnI-RV (SEQ ID NO: 25) listed in the Sequence Listing were used for PCR, for introduction of the NcoI site lying at the start methionine position and the KpnI recognition sequence at the 3'-end of the end codon. The amplified DNA fragments 15 were subcloned in pCR2.1 TOPO vector. After confirming lack of PCR-induced error, the DNA fragment cut out with NcoI and KpnI was linked to the NcoI and KpnI sites of pQE61, to obtain pSPB1642 as a pSPB1617cDNA E. coli expression vector. Ten different GT cDNA E. coli 20 expression vectors were constructed in the same manner. 1617BamHINcoI-FW SEQ ID NO: 24: 5'-GGG GGA TCC ATG GCT AGT GAG AGC CAA ATA-3' 1617XhoIKpnI-RW 25 SEQ ID NO: 25: 5'-CCC CTC GAG GGT ACC TCA CAA AAC ATT ATT CAC GAC-3' Each expression vector was introduced into E. coli JM109 (TOYOBO) and pre-cultured overnight at 37*C in LB medium containing ampicillin at a final concentration of 30 20 pg/ml. After adding 1 ml of pre-culturing solution to 50 ptg/ml of ampicillin and 50 ml of M9 medium containing 0.5% casamino acid and culturing to A600 = 0.6-1.0, IPTG (Isopropyl- -D-thiogalactopyranoside) was added to a final concentration of 0.1 mM and shake culturing was 35 carried out overnight at 27*C, and was followed by centrifugation at 3000 rpm for 10 minutes at 4 0 C to - 22 recover the cells. The cells were suspended in 10 ml of buffer solution (30 mM Tris-HCl pH 7.5, 30 mM NaCl), and after disruption of the E. coli with an ultrasonic treatment with a SONIFIER 250 (BRANSON), it was 5 centrifuged at 15,000 rpm, 10 min, 4*C and the obtained supernatant was used as the crude enzyme solution for the following activity assay. 3-2 Enzyme activity assay After loading THC (500 jg/ml ethanol solution) into 10 1 ml of the reverse-phase resin TOYOPEARL HW-40F (TOSOH) equilibrated with H 2 0, while diluting with H 2 0, it was rinsed with water to obtain THC substrate fixed on the resin. To 100 pl of the resin-fixed THC there were added 200 Rl of the crude enzyme solution obtained in 3-1 and 15 10 il of 5 mM UDP-glucose, and reaction was conducted at 300C for 1 hour. After removing the supernatant by centrifugation, the precipitated resin was rinsed with water and suspended in 300 pl of 50% acetonitrile containing 0.1% TFA (Trifluoroacetic acid), and the 20 flavonoids were freed from the resin by ultrasonic treatment. Upon centrifugation at 15,000 rpm, 5 min, 4*C, the insoluble portion of the obtained supernatant was removed with a filter (pore size: 0.45 mm, 4 mm Millex LH, Millipore), and the supernatant was analyzed by high 25 performance liquid chromatography (HPLC). The analysis conditions for chalcones and their glucosides were as follows. The column used was a Develosil C-30-UG-5 (4.5 mm* x 150 mm, Nomura Chemical Co., Ltd.), with a mobile phase 30 of H 2 0 containing 0.1% TFA as solution A and 90% acetonitrile containing 0.1% TFA as solution B, and after elution for 10 minutes with a linear concentration gradient from 20% solution B to 70% solution B, it was held for 5 minutes with 70% solution B. The flow rate 35 was 0.6 ml/min, and detection was carried out based on absorbance at 360 nm and the absorption spectrum from - 23 250-400 nm using an SPD-M6A PDA detector (Shimadzu Corp.). Elution of THC at a retention time of 10.7 min, and 2'-glucoside and 4'-glucoside at 8.5 min under these conditions was confirmed using samples of THC and THC 2' 5 and 4'-glucosides. Upon reaction of the pSPB1642-expressing E. coli extract, a new product in addition to the THC substrate was detected, eluting at 8.5 min. Since these were not detected with reaction using crude extract prepared in 10 the same manner from E. coli expressing only pQE61 vector or boiled crude extract of E. coli expressing pSPB1642, it was concluded that the product was produced by GT expressed from pSPB1642. The structure of the product was also examined by 'H-NMR analysis. The analysis was 15 performed using a JNM-EX400 (JEOL), under conditions as described in the literature (Plant Physiology 132, 1652 (2003)). As a result, the THC glucoside produced by the expression product of pSPB1642 was demonstrated to be THC 2'-glucoside. Thus, it was concluded that the cDNA 20 expressed by pSPB1642, i.e. pSPB1617 cDNA, codes for a protein with 2'CGT activity. Example 4. 4'CGT gene screening 2 The approximately 300,000 clones of the yellow Antirrhinum majus flower petal cDNA library were re 25 screened using the full-length pSPB1617 cDNA as the probe. The probe labeling by PCR was conducted in the same manner as described in Example 2, using 1617-F (SEQ ID NO: 26) and 1617-R (SEQ ID NO: 27) primers. The screening and nucleotide sequence analysis methods were 30 also the same as in Example 2. 1617-F SEQ ID NO: 26: 5'-ATG GGA GAA GAA TAC AAG AAA ACA-3' 1617-R SEQ ID NO: 27: 5'-TAA AAT TTG GTA GTT AAA CCG ATG 35 TA-3' As a result, five novel GT genes were obtained: pSPB1721, 1724, 1723, 1719 and 1725. The respective - 24 sequences are listed in the Sequence Listing (SEQ ID NO: 28-31 and 1). Among these, pSPB1725 cDNA contained a 1374 bp translation region (excluding the stop codon) coding for 5 a 50.8 kDa molecular weight protein composed of 457 amino acids, with an isoelectric point of 6.82. The amino acid sequence (SEQ ID NO: 2) encoded by the pSPB1725 cDNA was compared with the previously reported GT amino acid sequence, and only showed 14% homology with Livingstone 10 Daisy GT (Plant J. 19, 509 (1999)), 18% homology with perilla 5GT, 18% homology with perilla 3GT, 23% homology with gentian 3'GT and 31% homology with the amino acid sequence of the protein encoded by pSPB1617 used as the probe. The software used for homology analysis was 15 ClustalW included with MacVector ver.6.5.3 (Oxford Molecule), with parameters of Matrix Blosum 30, ketuple: 1, Gap penalty: 3, Topdiagonals: 5, Windows Size: 5. Example 5. Expression of obtained cDNA in E. coli 5-1. Expression vector construction 20 The five cDNAs obtained in Example 4 were examined by assaying the enzyme activity of the protein encoded by each cDNA using an E. coli expression system. The expression vector construction and expression method and activity assay method were the same as in Example 3. For 25 pSPB1725, for example, PCR reaction was conducted using the following two primers 1725-NcoI (SEQ ID NO: 32) and 1725-KpnI (SEQ ID NO: 33) for introduction of the NcoI recognition sequence at the 5'-end of the start codon. 1725-NcoI 30 SEQ ID NO: 32: 5'-CCC ATG GGA GAA GAA TAC AAG AAA-3' 1725-KpnI SEQ ID NO: 33: 5'-GGT ACC TAT AAA ATT TGG TAG TTA AA-3' The PCR solution (25 pl) consisted of 10 ng pSP1725 35 DNA, 1x ExTaq buffer (Takara), 0.2 mM dNTPs, 0.2 pmol/pl each of the 1725-NcoI and 1725-KpnI primers and 1.25 U ExTaq polymerase.

- 25 Reaction was conducted at 94 0 C for 5 minutes, followed by 28 cycles of reaction at 94 0 C for 1 minute, 55 0 C for 1 minute and 72'C for 2 minutes, and then by final treatment at 72*C for 7 minutes. The obtained PCR 5 product was subcloned in pCR2.1 TOPO vector (INVITROGEN) according to the method recommended by the manufacturer. Upon confirming the nucleotide sequence of the amplification product, the approximately 1.4 kb fragment cut out from pCR2.1 TOPO vector by NcoI and KpnI 10 treatment was linked to the NcoI and KpnI sites of pQE61 (QIAGEN) to obtain the E. coli expression vector pSPB1768. This was then introduced into E. coli JM109 (TOYOBO). E. coli expression vectors were also constructed for the other four cDNAs in the same manner 15 using pQE61, and were introduced into JM109. 5-2 Recombinant protein expression in E. coli and GT activity assay The E. coli transformants obtained in Example 5-1 were cultured under the same conditions as Example 3 and 20 used for activity assay of the proteins encoded by each cDNA. As a result, a peak attributed to THC glucoside was detected in the reaction product between THC and the crude enzyme solution of pSPB1768-containing E. coli. In order to identify the THC glucoside in greater detail, 25 HPLC analysis was again performed under conditions for separation of THC 2'-glucoside and THC 4'-glucoside, as follows. The column used was a YMC-ODS-A312 (6 mm4 x 150 mm, YMC Corp.), and with a mobile phase of H 2 0 containing 2% 30 acetic acid as solution A and methanol as solution B, for elution for 15 minutes with a linear concentration gradient from 15% solution B to 40% solution B and holding for 5 minutes with 40% solution B, followed by elution for 10 minutes with a linear concentration 35 gradient from 40% solution B to 62% solution B and holding for 2 minutes with 62% solution B. The flow rate - 26 was 1.0 ml/min. Detection was carried out based on absorbance at 360 nm and the absorption spectrum from 250-400 nm using an SPD-M6A PDA detector (Shimadzu Corp.). 5 Under these conditions, THC elutes at a retention time of 26.7 min, THC 2'-glucoside elutes at 19.8 min.and 4'-glucoside elutes at 20.6 min. The THC glucoside' found in the pSPB1768-expressing E. coli extract and THC reaction solution was believed to be 4'-glucoside because 10 it eluted at 20.6 min by analysis under these conditions. Since it was not detected upon reaction of crude extract prepared in the same manner from E. coli expressing only pQE61 vector, it was presumably the product of GT encoded by pSPB1725. These results confirmed that GT encoded by 15 pSPB1725 cDNA exhibits activity of transferring glucose to the 4'-hydroxyl group of THC. A new peak for elution at 15.5 min was also detected in addition to THC 4'-glucoside in the reaction solution. This substance exhibited the absorption spectrum of 20 naringenin, and matched the retention time for a naringenin 7-glucoside standard. Thus, the product that eluted at 15.5 min was believed to be naringenin 7 glucoside produced by post-glucosylation ring closure of THC 4'-glucoside produced by 4'CGT encoded in pSPB1725, 25 or naringenin 7-glucoside produced by the action of 4'CGT on naringenin produced by ring closure of THC. Example 6. Expression analysis of 4'CGT gene in Antirrhinum majus flower petals The expression profile of the 4'CGT gene encoded by 30 pSPB1725 in yellow Antirrhinum majus flower petals was analyzed by RT-PCR. Flower petals of yellow Antirrhinum majus (Butterfly Yellow variety) which accumulate aurones were separated at 5 growth stages. The five stages are, in order of youth, stages 1 (budding petal length of 1 35 cm), 2 (budding petal length of 1.0-1.5 cm), 3 (budding petal length of 1.5-2.0 cm), 4 (petal length of 2.0-2.5 cm, just prior to blooming) and 5 (petal length of >2.5 - 27 cm, bloomed petals), where stage 5 corresponds to the mature flower petal. The RNA was extracted from 1 g of separated flower petals, using an RNeasy Plant Mini Kit (QIAGEN). Reverse 5 transcription reaction was conducted using 1 ptg of obtained RNA as template to obtain cDNA. A SuperScript First-Strand Synthesis System for RT-PCR (GIBCO BRL) was used for the cDNA synthesis, and the synthesis conditions were according to the conditions recommended by the 10 system manufacturer. PCR was performed using the cDNA obtained at each stage as template and the 1725-NcoI (SEQ ID NO: 32) and 1725-KpnI (SEQ ID NO: 33) described in Example 5 as primers. For comparison between 4'CGT gene expression and endogenous gene expression, the 15 Antirrhinum majus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (SEQ ID NO: 34) (Nature 339, 46 (1989)) was used as the internal standard gene, and primers AmGAPDH-F (SEQ ID NO: 35) and AmGAPDH-R (SEQ ID NO: 36) were synthesized for amplification of this gene. 20 Also, primers AmAS-F (SEQ ID NO: 37) and AmAS-R (SEQ ID NO: 38) were synthesized for amplification of the Antirrhinum majus AS gene, as a comparison gene. AmGAPDH-F SEQ ID NO: 35:5'-TGT TGC TGT TAA CGA TCC AT-3' 25 AmGAPDH-R SEQ ID NO: 36:5'-AGC TCT TCC ACC TCT CCA-3' AmAS-F SEQ ID NO: 37:5'-ATG TTC AAA AAT CCT AAT ATC CGC-3' AmAS-R 30 SEQ ID NO: 38:5'-TTA GCC ATC AAG CTC AAT CTT GAC A 3' The PCR conditions were 12 cycles of 940C for 1 minute, 55*C for 1 minute and 72*C for 2 minutes, with the same reaction composition as in Example 3. The PCR 35 product was separated by 1% agarose gel electrophoresis, and then blotted on a Hybond-N nylon membrane (Amersham) - 28 according to ordinary protocol for detection of amplification product by hybridization. The hybridization method employed the previously mentioned non-radioisotope DIG-nucleic acid detection system, DIG 5 DNA labeling and detection kit, and was carried out according to the method recommended by the manufacturer. The probes used were cDNA for 4'CGT encoded in Antirrhinum majus AS, GAPDH and pSPB1725, and DIG labeling was accomplished using primers specific for each 10 gene (SEQ ID NO: 32, 33, 35-38) in the same manner as Example 2. As a result, both the 4'CGT and AS genes reached expression peaks at stage 4, thus exhibiting similar expression patterns with time. The expression patterns 15 for both genes also did not appear inconsistent with the accumulation patterns for chalcone 4'-glucosides and aurones in yellow Antirrhinum majus flower petals (Plant Sci. 160, 229 (2001)). These results suggest that expression of the 4'CGT 20 gene encoded in pSPB1725 is under the same control as the AS gene in Antirrhinum majus flower petals, and that both are involved in the same synthesis pathway, namely the synthesis pathway of aurone. Example 7. Co-expression of 4'CGT and AS in plant 25 7-1 Construction of 4'CGT expression cassette Plasmid pBE2113-GUS (Plant Cell Physiol. 37, 49 (1996)) was digested with SnaBI and religated to remove the omega sequence, and the obtained plasmid was designated as pUE6. Separately, plasmid pUCAP (van 30 Engelen et al. Transgenic Research 4,. 288-290, 1995) was digested with AscI and the ends blunted, after which PacI linker was inserted to prepare a plasmid designated as pUCPP. A fragment of pUE6 from the E1 2 35S promoter to the NOS terminator was inserted at the HindIII and EcoRI 35 sites of pUCPP to obtain plasmid pSPB540. The GUS gene portion of pSPB540 was replaced with the 4'CGT cDNA fragment cut out from pSPB1725, and the obtained plasmid N:\Melbourne\Cases\Patent\61000-61999\P61155.AU\Specis\Speci.doc 5 July 2007 - 29 was designated as pSFL203. That is, pSFL203 comprises pUCPP as the vector and has the 4'CGT expression cassette controlled by E1 2 35S promoter and NOS terminator. 7-2 Construction of AS expression cassette 5 Antirrhinum majus-derived AS cDNA (Science 290, 1163, (2000)) was inserted at the EcoRI and XhoI sites of pBluescript II SK-vector (Stratagene) to obtain a plasmid designated as pSPB251. An AS expression construct having the MacI promoter, the AS cDNA fragment cut out from 10 pSPB251 and the MAS terminator linked in plasmid pBINPLUS (van Engelen et al. Transgenic Research 4, 288-290, 1995) was designated as pSPB1624. 7-3 Creation of 4'CGT and AS co-expression construct The pSFL203 of 7-1 was cleaved with PacI and the 15 chalcone glucosylating enzyme gene expression cassette was cut out and inserted at the PacI site of plasmid pSPB1624 from 7-2. The obtained construct was designated as pSFL201. Thus, pSFL201 is designed so as to constitutively express the 4'CGT and AS genes when 20 introduced into plant cells. Example 8 Co-expression of 4'CGT and AS and inhibition of torenia DFR in plants 8-1 Construction of torenia-derived DFR gene expression-inhibiting cassette 25 The cDNA for torenia dihydroflavonol reductase (DFR) was obtained in the manner described in the literature (Plant Science 153, 33, 2000). The torenia DFR cDNA was linked with the vector pBluescriptII SK- to obtain a plasmid designated as pTDF10. This was used as a 30 template for PCR in the manner described in Example 3, using M13 reverse primer (SEQ ID NO: 39) from the vector sequence and the primer ThDFR-NcoI (SEQ ID NO: 40) having an NcoI recognition site introduced therein by nucleotide substitution in the torenia DFR cDNA sequence. The 35 approximately 0.75 kb fragment that was obtained was cloned in pCR2.1-TOPO (Invitrogen), and after confirming the nucleotide sequence, the 0.75 kb torenia DFR cDNA - 30 sequence was cut out with SacI and NcoI. Also, plasmid pTDF10 was cleaved with BamHI and NcoI, and a fragment containing 1.1 kb was recovered from the 5' end of torenia DFR cDNA. Separately, plasmid 5 pUCAP described in 7-1 was digested with PacI and its ends blunted, after which an AscI linker was inserted to obtain a plasmid designated as pUCAA. A fragment including E1 2 35S promoter-GUS-NOS terminator cut out from pUE6 was inserted at the HindIII and EcoRI sites of 10 pUCAA, and the obtained plasmid was designated as pSPB541. Plasmid pSPB541 was cleaved with BamHI and SacI, the GUS gene portion was removed, and there were inserted therein the 0.75 kb fragment and 1.1 kb fragment from torenia DFR cDNA, ligated in a direction linking the 15 NcoI sites of both fragments. Plasmid pSFL314 obtained in this manner, when introduced into a plant, can transcribe double-stranded RNA from the torenia DFR cDNA sequence under the control of E1 2 35S promoter, thus inhibiting expression of the torenia DFR gene by RNAi. 20 M13 reverse primer SEQ ID NO: 39: 5'-AACAGCTATGACCATG-3' ThDFR-NcoI SEQ ID NO: 40: 5'-GCTTTACCATGGAGTAATGAGCTT-3' 8-2 Co-expression of 4'CGT and AS and creation of 25 construct for inhibition of torenia DFR gene expression An XhoI linker was inserted upstream of the NOS terminator of pUE6 described in 7-1. The plasmid was digested with BamHI and XhoI to obtain a fragment comprising the E1 2 35S promoter-vector-NOS terminator, and 30 this fragment was ligated with the AS cDNA fragment cut out from pSPB215 with BamHI and XhoI as described in 7-2 to obtain plasmid pSPB211. The AS expression cassette was cut out from pSPB211 with HindIII and EcoRI and inserted at the HindIII and EcoRI sites of pBINPLUS. The 35 4'CGT expression cassette obtained by PacI cleavage of pSFL203 described in 7-1 was inserted at the PacI site of the plasmid obtained in this manner, to obtain plasmid - 31 pSFL304 having the 4'CGT and AS expression cassettes linked in tandem. Also, the torenia DFR double-stranded RNA transcription cassette described in 8-1 was inserted at the AscI site of pSFL304 to obtain plasmid pSFL307. 5 That is, pSFL307 includes three cassettes, for 4'CGT and AS expression and torenia DFR gene expression inhibition. Example 9 Co-expression of 4'CGT and AS and inhibition of torenia F3H gene expression in plant 9-1 Cloning of torenia F3H cDNA and construction of 10 expression inhibition cassette for the gene F3H cDNA obtained from perilla (Plant Mol Biol., 35, 915 (1997)) was used as a probe to obtain cDNA coding for the same enzyme in torenia. Specifically, a torenia cDNA library (Molecular Breeding, 6, 239, 2000) of 15 approximately 200,000 phage was screened in the same manner as Example 2 to obtain the torenia F3H cDNA listed as SEQ ID NO: 41. The torenia F3H cDNA was linked with the vector pBluescriptII SK- to obtain a plasmid designated as pSPB266. This was used as template for PCR 20 in the manner as Example 3, using M13 reverse primer (SEQ ID NO: 39) from the vector sequence and the primer ThF3H SalI-1 (SEQ ID NO: 42) having a SalI recognition site inserted therein by nucleotide substitution in the torenia F3H cDNA sequence. 25 The approximately 0.9 kb fragment that was obtained was cloned in pCR2.1-TOPO (Invitrogen), and the nucleotide sequence was confirmed. In the same manner, the primer ThF3H-SalI-2 (SEQ ID NO: 43) having a SalI recognition site inserted therein by nucleotide 30 substitution in the torenia F3H cDNA sequence and M13 reverse primer were used to prepare an approximately 0.75 kb DNA fragment which was cloned in pCR2.1-TOPO, and the nucleotide sequence was confirmed. Plasmid pSPB541 described in Example 8-1 was cleaved with BamHI and SacI, 35 the GUS gene portion was removed, and there were inserted therein a 0.9 kb fragment cut out from pCR2.1-TOPO by cleavage with BamHI and SalI and a 0.7 kb fragment cut - 32 out from pCR2.1-TOPO by cleavage with SacI and SalI, ligated in a direction linking the SalI sites of both fragments. Plasmid pSFL313 obtained in this manner, when introduced into a plant, transcribes double-stranded RNA 5 derived from the torenia F3H cDNA sequence under the control of E1 2 35S promoter, thus inhibiting expression of the torenia F3H gene by RNAi. ThF3H-SalI-1 SEQ ID NO: 42: 5'-TTCTCTGTCGACGCCCATTGCC-3' 10 ThF3H-SalI-2 SEQ ID NO: 43: 5'-CGCCGTGTCGACTCGCTTGAAG-3' 9-2 Co-expression of 4'CGT and AS and creation of construct for inhibition of torenia F3H gene expression The torenia F3H RNAi cassette was cut out from the 15 pSFL313 described in 9-1 by cleavage with AscI and inserted at the AscI site of pSFL304 described in Example 8-2 to obtain plasmid pSFL308. That is, pSFL308 includes three cassettes, for 4'CGT and AS expression and torenia F3H gene expression inhibition. 20 Example 10. Gene expression and flower color analysis in plant Plasmids pSFL201, pSFL307 and pSFL308 described in Example 7-9 were introduced into torenia (variety: Summerwave Blue (Suntory Flowers Ltd.)) by a publicly 25 known method. The transformation method was based on the method described in Mol. Breeding. 6, 239, (2000). Individuals exhibiting selection marker resistance were selected out and their flower colors observed. Among the pSFL201-introduced varieties, 22 of the 35 transformant 30 lines exhibited altered flower color with respect to the host, displaying a yellowish blue or yellowish gray color. However, no pure yellow color was found. Among the pSFL307-introduced varieties, 19 of the 36 transformant 35 lines exhibited altered flower color with respect to the host. Also, 6 of the 19 lines with altered flower color displayed almost pure yellow flower colors virtually - 33 without any tint of the original blue color of the host. Among the pSFL308-introduced varieties, 24 of the 39 transformant lines exhibited altered flower color with respect to the host. Also, 17 of the 24 lines with 5 altered flower color displayed almost pure yellow flower colors virtually without any tint of the original blue color of the host. Pigment analysis was performed on the lines with relatively notable flower color alteration. 10 The flower petals of the host variety and the transformants were immersed with 50% acetonitrile containing 0.1% trifluoroacetic acid (TFA) for extraction of the flavonoids, and then the aureusidin 6-glucosides and anthocyanidins were analyzed by high performance 15 liquid chromatography (HPLC). For anthocyanidin analysis, the flavonoids extracted from the flower petals were dissolved in 6N HCl and hydrolyzed by holding in boiling water for 20 minutes, after which the flavonoids were re-extracted with amyl alcohol and supplied for 20 analysis. The HPLC conditions were as follows. First, detection of AU 6-glucoside was performed using a SHIM-PACK FC-ODS column (50 x 4.6 mm, Shimadzu Corp.), and for the reverse phase, H 2 0 containing 0.05% TFA was used as solution A and acetonitrile containing 25 0.05% TFA was used as solution B. Elution for 3 minutes with a linear concentration gradient from 10%-23% solution B was followed by 17 minutes with 23% solution B, and then elution for 2 minutes with a linear concentration gradient from 23%-80% solution B was 30 followed by 3 minutes with 80% solution B. Elution was then performed for 2 minutes with a concentration gradient from 80%-10% solution B. The flow rate was 0.8 ml/min. Detection was carried out based on absorbance at 360, 400 nm and the absorption spectrum from 250-500 nm 35 using an SPD-MlOAVP PDA detector (Shimadzu Corp.). Under these conditions, THC 4'-glucoside and AU 6-glucoside samples elute at retention times of 14.17 min and 6.19 - 34 min, respectively. The column used for anthocyanidins was a YMC-ODS-A A312 (6 x 150 mm, YMC Corp.). For the reverse phase there was used a mixture of acetic acid, methanol and H 2 0 5 at 60:70:270, maintained for 11 minutes. Detection was carried out based on absorbance at 520 nm and the absorption spectrum from 400-600 nm using an SPD-M10AVP PDA detector (Shimadzu Corp.). Under these conditions, malvidin elutes at a retention time of 9.12 min. 10 As a result, with the pSFL201-introduced transformants, products matching the retention times and absorption spectra of THC 4'-glucoside and AU 6-glucoside were confirmed to be produced in the flower petals at 0.02% and 0.05% respectively (W/W as fresh flower petal 15 weight). Because anthocyanidins native to the host were also present in the transformants, it may be assumed that the yellowish blue or gray colors observed in the transformants were due to the copresence of anthocyanidins such as malvidin with the THC 4'-glucoside 20 and AU 6-glucoside. On the other hand, with the pSFL307- and 308 introduced transformants, products matching only the retention times and absorption spectra of the aurone AU 6-glucoside were confirmed to be produced in the flower 25 petals at 0.09% (W/W as fresh flower petal weight) in both cases. In the pSFL307- or pSFL308-introduced lines, it was confirmed that anthocyanidins native to the host were notably reduced to 10-50% of those anthocyanidins in the host flower petals. 30 Example 11. Confirmation of 4'CGT gene introduction by genomic Southern hybridization Lines among the transformants obtained in Example 10 which had relatively large accumulation of THC 4' glucoside and AU 6-glucoside based on the flower petal 35 pigment analysis were selected (3 lines from each construct-introduced variety) and subjected to genomic hybridization. A Phytopure Plant DNA Extraction kit - 35 (Amersham) was used for extraction of genomic DNA from approximately 1 g of transformant leaves, according to the method recommended by the manufacturer. Twenty pg of the obtained genomic DNA was cleaved with restriction 5 endonuclease KpnI, and after separation by 0.7% agarose gel electrophoresis and transfer to a Hybond-N+ nylon membrane according to ordinary protocols, a non radioisotope DIG-nucleic acid detection system was used for hybridization. 10 The methods of DIG labeling, hybridization and detection of the 4'CGT gene probes were according to the methods recommended by the manufacturer, as in Example 6. The hybridization results are shown in Fig. 2. Judging from restriction enzyme maps for pSFL201, pSFL307 and 15 pSFL308, it is possible to estimate the number of 4'CGT gene copies introduced into each transformant based on the number of bands detected by genomic Southern hybridization. As one band was observed for all of the pSFL201 20 introduced lines, it is deduced that each has one copy of the introduced gene. Individuals of line Nos. 2 and 4 among the pSFL307-introduced lines had 1 copy, while individuals of line No. 13 exhibited 2 bands and therefore presumably have introduced 2 copies of 4'CGT 25 cDNA. Since one band was observed for all of the pSFL308-introduced lines, it is deduced that each has one copy of the introduced gene. Example 12. Introduced gene expression analysis by quantitative RT-PCR 30 An RNeasy Plant Mini Kit (QIAGEN) was used for extraction of total RNA from buds of the stock variety and each of the transformant lines, cDNA was synthesized from 1 ptg of the obtained total RNA using a SuperScript TM First-Strand Synthesis System for RT-PCR (Invitrogen). 35 Using 1 il of the obtained cDNA as template, expression of torenia DFR and F3H and transcription products of the - 36 exogenous genes AS and 4'CGT was quantitatively analyzed with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Using the manufacturer's recommended software "Primer Express", there were designed 5 oligoprimers for specific amplification of each gene, and specific hybridizing TaqMan probes fluorescently labeled at both ends, and these were used for reaction. Quantitation of expression was carried out using the oligoprimers of SEQ ID NO: 54 and 55 and the TaqMan probe 10 of SEQ ID NO: 56 for torenia DFR, the oligoprimers of SEQ ID NO: 57 and 58 and the TaqMan probe of SEQ ID NO: 59 for torenia F3H, the oligoprimers of SEQ ID NO: 60 and 61 and the TaqMan probe of SEQ ID NO: 62 for AS and the oligoprimers of SEQ ID NO: 63 and 64 and the TaqMan probe 15 of SEQ ID NO: 65 for 4'CGT. Torenia DFR SWB DFR-1158F 5'-AAT GGG ATG CTT CCG ACT TCT-3' (SEQ ID NO: 54) 20 SWB DFR-1223R 5'-CAG TGG TTT CTG CCA TTG CTT-3' (SEQ ID NO: 55) SWB DFR-1180T 5'-AGG AAA AAA CAG GCT GAA AA-3' (SEQ ID NO: 56) Torenia F3H 25 Torenia F3H-1035F 5'-CAT CGA GCG GTG GTG AAT T-3' (SEQ ID NO: 57) Torenia F3H-1101R 5'-CTG GCG ATG GGT TTT GAA A-3' (SEQ ID NO: 58) Torenia F3H-1055T 30 5'-AAA CAC GAA TAG AAT GTC G-3' (SEQ ID NO: 59) AS AmAS-1545F 5'-GAA GAT GAC CTT GCG GTG ATT T-3' (SEQ ID NO: 60) AmAS-1638R 35 5'-TTG TCC TCT TCC CCT TTA TAG GTT T-3' (SEQ ID NO: 61) AmAS-1582T - 37 5'-AGT TCG CCG GGA GTT TCG TGA GTC TG-3' (SEQ ID NO: 62) 4'CGT AmGTcgl2-908F 5 5'-GGT TGG CCC GCA TTT CA-3' (SEQ ID NO: 63) AmGTcgl2-966R 5'-TAG AAA ACC CTC CGG CAG AA-3' (SEQ ID NO: 64) AmGTcgl2-929T 5'-AGA TGG ACT TAA ATG CG-3' (SEQ ID NO: 65) 10 As an endogenous control there was used torenia glyceraldehyde phosphate dehydrogenase (GAPDH). The oligoprimers used were SWB GAPDH-794F (5'-GCA TTG AGC AAG ACG TTT GTG-3') (SEQ ID NO: 66) and SWB GAPDH-859R (5' ACG GGA ACT GTA ACC CCA TTC-3') (SEQ ID NO: 67), and the 15 TaqMan probe used was SWB GAPDH-816T (5'-AGC TTG TGT CGT GGT ACG-3') (SEQ ID NO: 68). The reaction solution was prepared to a total volume of 50 p.l, comprising the cDNA of each transformant line, 1 x TaqMan Universal Master Mix (Applied Biosystems), 100 20 nM of each oligoprimer and 100 nM of TaqMan probe. Reaction was conducted at 50*C for 2 minutes and 95*C for 10 minutes, followed by 40 cycles of reaction at 95*C for 15 seconds, 60*C for 1 minute, and the production of PCR amplification product was detected in real time. As a 25 result, high expression was confirmed both for introduced AS and 4'CGT in the pSFL201-introduced lines. It was also confirmed that the introduced AS and 4'CGT were both expressed in the pSFL307-introduced lines, and that endogenous DFR mRNA was inhibited to about 10% compared 30 to the stock variety. In the pSFL308-introduced lines, it was confirmed that both the introduced AS and 4'CGT were expressed, while endogenous F3H mRNA was inhibited to about 5% compared to the stock variety. Example 13. Assay of 4'CGT glucosylating activity 35 on PHC PHC 4'-glucoside has been confirmed in yellow - 38 Antirrhinum majus flower petals (Sato, T., et al. Plant Sci. 160, 229-236 (2001)), and it is known that AS can produce bracteatin and bracteatin 6-glucoside using PHC and PHC 4'-glucoside as substrate. In order to determine 5 whether or not 4'CGT can catalyze PHC 4'-position .glucosylation reaction, the glucosylating activity of 4'CGT for PHC was assayed. Enzyme reaction was conducted according to Example 5-2, using recombinant 4'CGT expressed in E. coli and PHC fixed on resin by the same 10 method as Example 3-2 as substrate. The HPLC conditions were as follows. The column used was a YMC-ODS-A312 (6 mm# x 150 mm, YMC Corp.), with a mobile phase of H 2 0 containing 2% acetic acid as solution A and methanol as solution B, for 15 elution for 22 minutes with a linear concentration gradient from 15% solution B to 40% solution B and holding for 5 minutes with 40% solution B, followed by elution for 14 minutes with a linear concentration gradient from 40% solution B to 62% solution B and 20 holding for 2 minutes with 62% solution B. The flow rate was 1.0 ml/min. Detection was carried out based on absorbance at 360 nm and the absorption spectrum from 220-400 nm using an SPD-Ml0AP PDA detector (Shimadzu Corp.). Under these conditions, THC elutes at a 25 retention time of 38.2 min, THC 2'-glucoside elutes at 27.7 min, 4'-glucoside elutes at 30.0 min, PHC elutes at 32.4 min and PHC 4'-glucoside elutes at 24.3 min. The PHC glucoside found in the pSPB1768-expressing E. coli extract was identified as PHC 4'-glucoside because it 30 eluted at 24.3 min by analysis under these conditions. When PHC was reacted with a crude extract prepared in the same manner from E. coli expressing only pQE61 vector, no PHC glucoside was detected. Thus, PHC 4'-glucoside is presumably a product of GT encoded in pSPB1725. These 35 results confirmed that GT encoded in pSPB1725 cDNA exhibits activity of transferring glucose to the 4' hydroxyl group of PHC. The results described above - 39 demonstrated that 4'CGT catalyzes not only 4' glucosylation of THC but also 4'-glucosylation of PHC. Example 14. Functional analysis of 4'CGT and AS using torenia transformants 5 14-1 Creation of construct A 2.4 kb 4'CGT expression cassette portion was cut out from the pSFL203 described in Example 7 with PacI, and was inserted at the PacI site of pBINPLUS to obtain plasmid pSFL209. Plasmid pSFL209 expresses 4'CGT alone 10 in plants. A 2.7 kb F3H expression cassette was cut out from pSFL313 described in Example 9 with AscI, and this was inserted at the AscI site of pBINPLUS to obtain plasmid pSFL210. Plasmid pSFL210 is designed to transcribe 15 double-stranded RNA of the torenia F3H gene in torenia plants, thereby inhibiting expression of F3H. Separately, for AS, an AS cDNA fragment cut out from pSPB251 described in Example 7 with BamHI and XhoI was inserted at the BamHI and XhoI sites of pSPB120' 20 described in a patent application (P2003-293121), to obtain vector pSPB211 which expresses AS in plants. 14-2 RT-PCR expression analysis of introduced genes and flower color analysis Plasmids pSFL209, pSFL210 and pSFL211 described in 25 Example 14-1 were introduced into torenia (variety: Summerwave Blue (Suntory Flowers Ltd.)) by the method described in Example 10. The transformation method was based on the method described in Mol. Breeding. 6, 239, (2000). Individuals exhibiting selection marker 30 resistance were selected out. An RNeasy Plant Mini Kit (QIAGEN) was used for extraction of total RNA from buds of each line of the transformants and the stock variety Summerwave Blue, and cDNA was synthesized from 1 ig of the obtained total RNA by reverse transcription using a 35 SuperScriptTm First-Strand Synthesis System for RT-PCR (Invitrogen). RT-PCR reaction was also conducted using ExTaq (TaKaRa) according to the method recommended by the - 40 manufacturer. For amplification of AS mRNA there were used primers AmAS-INSITU-FW (5'-AATTATTTCCCAATGTTCAAAAAT 3') (SEQ ID NO: 44) and AmAS-INSITU-RV (5' TGGAGCTTTAGGTTTGTGAAA-3') (SEQ ID NO: 45), for 5 amplification of Antirrhinum majus 4'CGT mRNA there were used primers KIR-INSITU-FW (5'-ATGGGAGAAGAATACAAGAAAAC 3') (SEQ ID NO: 46) and KIR-INSITU-RV (5' TCTTACGATAAAACAAACTCA-3') (SEQ ID NO: 47), for amplification of endogenous F3H mRNA there were used 10 primers T.F3H-923F (5'-ATC ATC GAG CGG TGG TGA A-3') (SEQ ID NO: 48) and T.F3H-1339R (5'-TGG CCG ACT AGG CAA TAC AAT-3') (SEQ ID NO: 49), and for amplification of mRNA of GAPDH as the endogenous standard gene there were used primers T.GAPDH-F87 (5'-CCC TTC TGT TTG GTG AAA AGC C-3') 15 (SEQ ID NO: 50) and T.GAPDH-R692 (5'-CCT CGG ATT CCT CCT TGA TAG C-3') (SEQ ID NO: 51). As a result, of the 41 transformant lines obtained among the pSFL209-introduced lines, the introduced Antirrhinum majus 4'CGT transcripts were detectable in 37 lines, but no flower color 20 alteration was observed in any of the lines. Of the 44 transformant lines obtained among the pSFL210-introduced lines, significant decrease in the amount of endogenous F3'H transcript was detectable in 37 lines, and these lines exhibited white or mixed violet/white flower 25 colors. In addition, of the 41 transformant lines obtained among the pSPB211-introduced lines, expression of the introduced AS was confirmed in 31 lines, and no flower color alteration was observed in any of the lines. Pigment analysis was carried out for the pSFL209 30 introduced lines and pSPB211-introduced lines in which transcripts of the introduced genes were detected, and for the pSFL210-introduced lines that exhibited white flower color. The flower petals of Summerwave Blue and each of the transformants were wetted with 50% 35 acetonitrile containing 0.1% trifluoroacetic acid (TFA) for extraction of the flavonoids, and then AU6-glucoside and anthocyanidins were analyzed by high performance - 41 liquid chromatography (HPLC). Analysis of the anthocyanidins was carried out in the same manner as Example 10. The HPLC conditions were as follows. First, detection of AU6-glucoside was performed 5 using a SHIM-PACK FC-ODS column (50 x 4.6 mm, Shimadzu Corp.), and for the reverse phase, H 2 0 containing 0.05% TFA was used as solution A and acetonitrile containing 0.05% TFA was used as solution B. Elution for 3 minutes with a linear concentration gradient from 10%-23% 10 solution B was followed by 17 minutes with 23% solution B, and then elution for 2 minutes with a linear concentration gradient from 23%-80% solution B was followed by 3 minutes with 80% solution B. Elution was then performed for 2 minutes with a concentration 15 gradient from 80%-10% solution B. The flow rate was 0.8 ml/min. Detection was carried out based on absorbance at 360 and 400 nm and the absorption spectrum from 250-500 nm using an SPD-MlOAVP PDA detector (Shimadzu Corp.). Under these conditions, THC 4'-glucoside and AU6 20 glucoside samples elute at retention times of 14.17 min and 6.19 min, respectively. The column used for anthocyanidins was a YMC-ODS-A A312 (6 x 150 mm, YMC Corp.). For the reverse phase there was used a mixture of acetic acid, methanol and H 2 0 at 60:70:270, maintained 25 for 11 minutes. Detection was carried out based on absorbance at 520 nm and the absorption spectrum from 400-600 nm using an SPD-MlOAVP PDA detector (Shimadzu Corp.). Under these conditions, malvidin elutes at a retention time of 9.12 min. 30 As a result, with the pSFL209-introduced lines, a product matching the retention time and absorption spectrum of THC 4'-glucoside was confirmed to be produced at 0.036-0.762 mg per gram of fresh flower petal weight. Anthocyanidins naturally found in the host were also 35 present. The anthocyanidin levels in the pSFL210-introduced lines were reduced to about 1% of the anthocyanidin level - 42 in host flower petals. In the pSPB211-introduced lines, no alteration in flavonoid pigment was observed compared to the host. No product matching the retention times and absorption 5 spectra of aurones was detected in any of the transformants. This indicated that chalcone glucosides and aurones are not synthesized in plants merely by inhibition of F3H expression and overexpression of AS, but that aurones are 10 synthesized by co-expression of 4'CGT and AS. Overexpression of 4'CGT alone can accumulate chalcone 4' glucoside, for alteration in flower color. Example 15. Cloning of 4'CGT cDNA from Linaria bipartita 15 In the same manner as Example 1, RNA extracted from Linaria bipartita buds and bloomed flower petals was used to prepare a cDNA library. A library of 8.0 x 105 pfu/ml was obtained from the bud-obtained RNA, while a cDNA library of 1.0 x 106 pfu/ml was obtained using bloomed 20 flower petal cDNA. Screening was performed using approximately 3.0 x 105 pfu of phage from each library, with 4'CGT cDNA encoded in the Antirrhinum majus pSPB1725 described in Example 4 as the probe. The probe labeling, hybridization and 25 subsequent membrane rinsing and detection were carried out in the same manner as Example 2. Nineteen positive clones were finally obtained as a result. When nucleotide sequencing was performed on 8 of the cDNAs having lengths (about 1.5 kb) as expected for cDNA 30 encoding chalcone glucosyltransferase, the 8 clones all had identical sequences and the clone with the longest cDNA was designated as pSFL409. The nucleotide sequence of this cDNA is listed as SEQ ID NO: 69, and the amino acid sequence encoded by it is listed as SEQ ID NO: 70. 35 The amino acid sequence encoded by the pSFL409 cDNA was shown to have high homology with that of Antirrhinum majus chalcone 4'-glucosyltransferase. In comparison - 43 with Antirrhinum majus chalcone 4'-glucosyltransferase cDNA, however, the amino acid sequence encoded by pSFL409 cDNA was found to be incomplete cDNA lacking about 10 bp from the start methionine. Therefore, a Gene Racer RACE 5 kit (Invitrogen) was used for amplification of the upstream cDNA fragment including the putative start methionine by the 5'RACE method, and this was cloned in pCRII-TOPO vector and designated as plasmid pSFL417. Linaria bipartita cDNA including the full length showed 10 65% sequence identity with Antirrhinum majus 4' glucosyltransferase on the amino acid level. Example 16. Expression and activity assay of Linaria bipartita cDNA in E. coli The full-length Linaria bipartita cDNA was 15 introduced at the NcoI and KpnI sites of the E. coli expression vector pQE61, and activity of the protein encoded by this Linaria bipartita cDNA was analyzed with an E. coli expression system. First, for creation of the E. coli expression construct, PCR was carried out using 20 pSFL417 as template and primers 417-NcoI (CCCATATATAGCCATGGAAGATACCATCG) (SEQ ID NO: 52) and 409 EcoRI (TAGTGTTGTGGAGTCGGGGGATTTCG) (SEQ ID NO: 53). Thus, an NcoI site was inserted at the start methionine position of pSFL417, and an EcoRI site was inserted at 25 the 3'-end. The products of cleaving this with NcoI/EcoRI and cleaving pSFL409 cDNA with EcoRI/KpnI were cloned at the NcoI/KpnI site of the E. coli expression vector pQE61, to obtain an E. coli expression construct (pSFL418) comprising the full-length Linaria bipartita 30 cDNA. This was introduced into E. coli JM109 and the recombinant protein activity was assayed in the same manner as Example 12. Upon reaction of the pSFL418 possessing E. coli extract using THC as substrate, THC 35 4'-glucoside was detected with a retention time of 30.0 min. In separate reaction of pQE61-possessing E. coli extract with THC as a control experiment, absolutely no - 44 THC 4'-glucoside was detected. Glucosylation activity for PHC was also assayed in the same manner as Example 12. As a result, upon reaction of pSFL418-possessing E. coli extract, PHC 4'-glucoside was detected with a 5 retention time of 24.3 min. However, no PHC glucoside was detected in the control experiment. These results suggest that Linaria bipartita cDNA cloned in SFL418 codes for 4'CGT.

Claims (17)

1. An isolated gene coding for a protein having activity of transferring a sugar to the chalcone 4' 5 position, wherein said gene is selected from the group consisting of: (i) a gene coding for the amino acid sequence listed as SEQ ID NO: 2 or SEQ ID NO: 70; (ii) a gene which codes for a protein having the 10 amino acid sequence listed as SEQ ID NO: 2 or SEQ ID NO: 70 with a modification of one or a plurality of amino acids that are added, deleted and/or substituted with other amino acids, and having activity of transferring a sugar to the chalcone 4'-position; and 15 (iii) a gene which hybridizes to the nucleotide sequence listed as SEQ ID NO: 1 or SEQ ID NO: 69 under stringent conditions and codes for a protein having activity of transferring a sugar to the chalcone 4' position. 20

2. A gene according to claim 1, which is derived from the family Scrophulariaceae.

3. A gene according to claim 2, which is derived from Antirrhinum majus or Linaria bipartita.

4. A vector comprising a gene according to any one 25 of claims 1 to 3.

5. An isolated host cell transformed by a vector according to claim 4.

6. A protein encoded by a gene according to any one of claims 1 to 3. 30

7. A method of producing a protein having activity of transferring a sugar to the chalcone 4'-position, comprising culturing or growing a host cell according to claim 5 and obtaining said protein from said host cell.

8. A plant comprising a gene according to any one 35 of claims 1 to 3 introduced therein or a progeny of said plant having the same properties as said plant, or a vegetatively propagated plant or tissue from such a 1034894 _1 (GHMatters) 28-Oct-09 46 plant.

9. A flower cut from a plant according to claim 8.

10. A method for transferring a sugar to the chalcone 4'-position using a gene according to any one of 5 claims 1 to 3.

11. A plant comprising modified flower color obtained by introducing and expressing a gene according to any one of claims 1 to 3 into the plant, or a progeny of said plant having the same properties as said plant. 10

12. A plant according to claim 11, wherein the flower color has a yellow tint.

13. A method of altering a flower color to yellow, comprising the steps of introducing into a plant a gene according to any one of claims 1 to 3 together with a 15 gene coding for aureusidin synthase and expressing the genes in the plant.

14. A method of altering a flower color to yellow, comprising the steps of introducing into a plant a gene according to any one of claims 1 to 3 together with a 20 gene coding for aureusidin synthase, and expressing the genes in the plant, while also inhibiting expression of a flavonoid synthesis pathway gene in the plant.

15. A method of altering a flower color to yellow, comprising the steps of introducing into a plant a gene 25 according to any one of claims 1 to 3 together with a gene coding for aureusidin synthase, and expressing the genes in the plant, while also inhibiting expression of dihydroflavonol reductase gene in the plant.

16. A method of altering a flower color to yellow, 30 comprising the steps of introducing into a plant a gene according to any one of claims 1 to 3 together with a gene coding for aureusidin synthase, and expressing the genes in the plant, while also inhibiting expression of flavanone 3-hydroxylase gene in the plant. 35

17. A gene according to claim 1, a vector according to claim 4, a host cell according to claim 5, a protein according to claim 6, a plant according to claim 8 or 1034894 1 (GHMatters) 28-Oct-09 47 claim 11, a flower according to claim 9 or a method according to any one of claims 7, 10 or 13 to 16, substantially as hereinbefore described, with reference to the examples, and, or figures. 5 1034894_1 (GHMatters) 28-Oct-09